Cellulase mutants

Selective Screening Methods for the Isolation of High Yielding Cellulase Mutants of Trichoderma reesei... 

Figure 1. Genealogy of high-yielding cellulase mutants. Yield of cellulase in FP units/mL (42) under controlled fermentor conditions (=)=) and...

NG14. This mutant is capable of elaborating 15 FP units/mL with a productivity of 45 units/L/hr. The final concentration of soluble extracellular protein is 21.2 mg/mL. Samples of this enzyme preparation were sent to G. Pettersson at the University of Uppsala, Sweden, for quantification of each of the enzymes in the cellulase complex using purified antibodies to the individual enzymes. The quantitative antigen-antibody reaction showed that 600 mg/g of this enzyme preparation was one enzyme, cellobiohydrolase. This represents a yield of 13 g/L of cellobio-hydrolase, which is a 100-fold increase over the amount of cellobiohydrolase obtained with strain QM 9414 (130 mg/L) the best previously existing cellulase mutant (G. Pettersson, personal communication). 

Much effort has been expended over the years on increasing enzyme production of T. reesei by isolation of high yielding mutants and optimizing media and fermentation conditions. Strains have been isolated that produce 2-6 times the cellulase productivity of the parent wild strain (QM 6a) in batch culture. The mutants produce higher levels of cellulase protein but the specie activity of the enzymes and the proportions of the individual components (ca. 30% endo- -glucanase, 70% o- -glucanase, and less 1% cellobiase) are similar to those of the parent. 

Cellulase-Negative Xylanase-Positive Mutants. There are two reports concerning the selection of such mutants from filamentous fungi, one on Pofyporus adm-tus (32) and the other cmi Trichoderma reesei (Durand, H. et al. Society CAYLA, Toulouse, France, unpublished results). An analysis of the eliminated cellulase genes has not been done, so it is not known if the mutants negative in endo(l- 4)-p-glucan-ase were deficient also in cellobiohydrolases. 

One of the favored organisms for study of cellulolysis by Trichoderma is T. reesei. Consequently, many mutant strains which hyperproduce cellulase have been obtained by treatment with ultraviolet light, gamma irradiation, the linear accelerator, diethyl sulphate and N-methyl-N -nitro-N-nitroso-guanidine (7). Whereas much of the study of T. reesei has been with cellulose as substrate, it is relevant to consider the other fractions of natural lignocelluloses hemicellulose and holocellulose (the combined cellulose and hemicellulose fraction). 

Targonski, Z. and Pielecki, J., Continuous semi-solid cultivation for the production of cellulase by Trichoderma reesei mutants using a polyurethane foam carrier and a liquid medium, Acta-Biotechnol. 1995 vol. 15, no. 3, pp. 289-296. 

Targonski and Pielecki investigated the production of cellulase using immobilized mycelium of Trichoderma reesei mutants on polyurethane foam impregnated... 

However, in the polysaccharides obtained from some mutant strains, there are deviations from this idealized structure.44 Xanthan is relatively resistant to enzymic hydrolysis, but it has been cleaved by an enzyme preparation from a Bacillus sp. at moderate temperatures and in the presence of buffer salts, yielding mono- and oligo-saccharides 45 A partially purified, enzyme preparation46 hydrolyzed deacetylated or depyruvated xanthan, and also xanthan from several wild-type and mutant strains of Xanthomonas. The release of reducing material varied little with xanthan preparations having differences in O-acetyl and pyruvic acetal contents. Under similar conditions of incubation, cellulase acted only on xanthan from mutant strains that had defective side-chain formation. 

Component enzymes of the cellulase system have been purified from several microbial species (1-13), among which mutants of the imperfect fungus Trichoderma provide the highest levels of extracellular enzyme activity (14). From this organism have been purified / -glucosi-dases (EC 3.2.1.21), endo-l,4-/ -D-glucanases (EC 3.2.1.4) and 1,4-/ -d-... 

Montenecourt and Eveleigh (37), using a special agar screening technique, have also isolated a cellulase enhanced mutant (NG-14). They suggest, however, that the cellobiohydrolase and the endoglucanase biosynthesis are regulated by different controls. They base this assumption on data for the relative ratios of cellulases obtained from this mutant needed to hydrolyze different substrates. 

---